The energetic state of microbial cells is very important during cell growth and product production. Currently, significant numbers of valuable products are produced through anaerobic fermentation. However, productivity, mainly production rate, of these processes is usually hampered by slow cell growth and low culture density. This is because under anaerobic conditions the cell generates much less energy from the nutrient; it mostly derives its energy through glycolysis that only yields two moles of ATP per mole of glucose consumed. This ATP yield is much lower than that of aerobic respiration, however, the remaining carbon is conserved for product formation. Certainly, a higher energy yield through aerobic respiration can be achieved, but only at the expense of releasing carbon atoms stored in the feedstock (hence lower product yields).
The aerobic respiratory chain of Escherichia coli (E. coli) is composed of a number of membrane-bound, multisubunit enzymes located within the cytoplasmic membrane. Dehydrogenases such as NADH dehydrogenase or succinate dehydrogenase reduce ubiquinone to ubiquinol within the cytoplasmic membrane. Ubiquinol diffuses within the membrane bilayer and is oxidized by either of two quinol oxidase complexes: the cytochrome O complex or the cytochrome D complex. For a complete review of E. coli respiration, see Ingledew and Poole (1984) incorporated herein by reference.
Two operating procedures have been developed in an attempt to strike an ideal balance between energy generation through aerobic respiration (for better cell growth and cell “fitness”) and carbon conservation. One approach is to operate the bioreactor under microaerobic conditions. This reactor configuration provides a slight increase in ATP supply and limits the amount of carbon lost during aerobic respiration. However, it is technically difficult to maintain a constant microaerobic environment in real processes, especially for large bioreactors. Variation in extent of aeration is a problem even in those situations where near complete oxygen saturation is desired. Any increased supply of oxygen will favor cell growth and subsequently decrease the product yield.
A second strategy is to perform the process in two stages. The first stage is to grow the cell under aerobic conditions and then switch to anaerobic conditions for product formation. This strategy avoids the possibility of oxygen oversupply and guarantees a certain product yield for the second stage. However, the supply of ATP still is low during the product formation stage. There are also biological complexities that arise during the transition (proper synthesis and activation of required proteins and cofactors, triggering of cellular stress responses).
What is required to improve production in engineered bacterial cells is a balance between energy generation through aerobic respiration and carbon atom conservation by limited respiration. By limiting respiration, the cell will not “waste” carbon atoms even when the oxygen supply is abundant. At the same time, a sufficient supply of ATP will be available for cell growth and product formation throughout the fermentation processes. Strain respiration can be maintained at levels required for specific bioproduction needs, thus optimizing respiration and carbon conservation.